Characterization of the pathway for transport of the cytoadherence‐mediating protein, PfEMP1, to the host cell surface in malaria parasite‐infected erythrocytes

Abstract

SummaryThe Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of antigenically diverse proteins is expressed on the surface of human erythrocytes infected with the malaria parasite P. falciparum , and mediates cytoadherence to the host vascular endothelium. In this report, we show that export of PfEMP1 is slow and inefficient as it takes several hours to traffic newly synthesized proteins to the erythrocyte membrane. Upon removal by trypsin treatment, the surface-exposed population of PfEMP1 is not replenished during subsequent culture indicating that there is no cycling of PfEMP1 between the erythrocyte surface and an intracellular compartment. The role of Maurer's clefts as an intermediate sorting compartment in trafficking of PfEMP1 was investigated using immunoelectron microscopy and proteolytic digestion of streptolysin O-permeabilized parasitized erythrocytes. We show that PfEMP1 is inserted into the Maurer's cleft membrane with the Cterminal domain exposed to the erythrocyte cytoplasm, whereas the N-terminal domain is buried inside the cleft. Transfer of PfEMP1 to the erythrocyte surface appears to involve electron-lucent extensions of the Maurer's clefts. Thus, we have delineated some important aspects of the unusual trafficking mechanism for delivery of this critical parasite virulence factor to the erythrocyte surface.

A Cluster of Ring Stage–specific Genes Linked to a Locus Implicated in Cytoadherence inPlasmodium falciparumCodes for PEXEL-negative and PEXEL-positive Proteins Exported into the Host Cell

“…(B) Schematic depiction of the treatment of IRBCs (top cell) with SLO (second cell from top) or saponin (sap, bottom cell). SLO treatment releases only the cytoplasm of the IRBC (Ansorge et al, 1996;Kriek et al, 2003). Saponin treatment also permeabilizes the PVM (Ansorge et al, 1996) and the Maurer's clefts (this study).…”

“…This occurs concomitant with other extensive host cell modifications and involves export of a large number of parasite proteins into the RBC, causing dramatic changes in the topology, permeability, deformability and adhesiveness of the host cell (for reviews, see Cooke et al, 2001Cooke et al, , 2004Marti et al, 2005). PfEMP-1 occurs on the RBC surface ϳ16-20 h after invasion (Gardner et al, 1996;Kriek et al, 2003), coin- Address correspondence to: Tobias Spielmann (tobias.spielmann@ gmail.com) or Donald L. Gardiner (don.gardiner@qimr.edu.au).…”

“…Because the smaller bands present in the insoluble fraction were not visible in total extracts, it is possible that these smaller species resulted from nonphysiological limited proteolysis in this fraction (which must have occurred despite the presence of protease inhibitors). To biochemically confirm the IFA results and to analyze the orientation of REX2 in the Maurer's clefts, we treated IRBCs with SLO, which permeabilizes the RBC membrane of IRBCs but leaves the PVM (Ansorge et al, 1996) and the Maurer's clefts intact (Kriek et al, 2003; Figure 5B). These cells were then subjected to digestion with proteinase K in the presence or absence of saponin and analyzed by immunoblotting using the serum raised against REX2, which is directed against its C-terminal region.…”

“…This occurs concomitant with other extensive host cell modifications and involves export of a large number of parasite proteins into the RBC, causing dramatic changes in the topology, permeability, deformability and adhesiveness of the host cell (for reviews, see Cooke et al, 2001Cooke et al, , 2004Marti et al, 2005). PfEMP-1 occurs on the RBC surface ϳ16-20 h after invasion (Gardner et al, 1996;Kriek et al, 2003), coin- Address correspondence to: Tobias Spielmann (tobias.spielmann@ gmail.com) or Donald L. Gardiner (don.gardiner@qimr.edu.au).Abbreviations used: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IRBC, infected red blood cell; KAHRP, Knob-associated histidine-rich protein; MAHRP, membrane-associated histidine-rich protein; PEXEL, Plasmodium export element; PfEMP, Plasmodium falciparum erythrocyte membrane protein; PV, parasitophorous vacuole; PVM, parasitophorous vacuole membrane; RBC, red blood cell; REX, ring-exported protein; SBP, skeleton-binding protein; SERP, serine-rich protein; VTS, vacuolar transport signal. ciding with the end of the ring stage.…”

Development and host cell modifications of Plasmodium falciparum blood stages in four dimensions

“…During this phase, Maurer's clefts change from a mobile to a fixed state, and a very mild echinocytosis of the host cell occurs. Transition to the trophozoite coincides temporally with the appearance of the cytoadherence protein PfEMP1 on the host cell surface 52 , leading to sequestration in vivo 10 . The echinocytosis might therefore indicate processes of surface remodelling that affect the homeostasis of the erythrocyte or changes to the host cell cytoskeleton.…”